Dynamic changes in chromatin accessibility and nucleosome positioning in the control of antigen receptor rearrangement
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE132171 | GEO | 2019/06/05
REPOSITORIES: GEO
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Project description:Chromatin looping mediated by the CCCTC binding factor CTCF regulates V(D)J recombination at antigen receptor loci. CTCF-mediated looping can influence recombination signal sequence accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate through-space interactions between recombination signal sequences, independent of the RAG recombinase. However, in all prior instances in which CTCF-mediated looping was shown to influence V(D)J recombination, it was not possible to fully resolve the relative contributions to the V(D)J recombination phenotype of changes in accessibility, RAG-tracking, and RAG-independent long-distance interactions. Here, to assess mechanisms by which CTCF-mediated looping can impact V(D)J recombination, we introduced an ectopic CTCF binding element (CBE) immediately downstream of Eδ in the murine Tcra-Tcrd locus. The ectopic CBE impaired inversional rearrangement of Trdv5 in the absence of measurable effects on Trdv5 transcription and chromatin accessibility. Moreover, although the ectopic CBE limited directional RAG tracking from the Tcrd recombination center, such tracking cannot account for Trdv5-to-Trdd2 inversional rearrangement. Rather, the defect in Trdv5 rearrangement could only be attributed to a reconfigured chromatin loop organization that limited RAG-independent through-space interactions between the Trdv5 and Trdd2 RSSs. We conclude that CTCF can regulate V(D)J recombination by segregating RSSs into distinct loop domains and inhibiting RSS synapsis, independent of any effects on transcription, RSS accessibility and RAG tracking. RAG-initiatd Tcrd D segment rearrangements in developing thymocytes were generated by deep sequencing using illumine Miseq
2016-09-02 | E-GEOD-81351 | biostudies-arrayexpress
Project description:Chromatin looping mediated by the CCCTC binding factor CTCF regulates V(D)J recombination at antigen receptor loci. CTCF-mediated looping can influence recombination signal sequence accessibility by regulating enhancer activation of germline promoters. CTCF-mediated looping has also been shown to limit directional tracking of the RAG recombinase along chromatin, and to regulate through-space interactions between recombination signal sequences, independent of the RAG recombinase. However, in all prior instances in which CTCF-mediated looping was shown to influence V(D)J recombination, it was not possible to fully resolve the relative contributions to the V(D)J recombination phenotype of changes in accessibility, RAG-tracking, and RAG-independent long-distance interactions. Here, to assess mechanisms by which CTCF-mediated looping can impact V(D)J recombination, we introduced an ectopic CTCF binding element (CBE) immediately downstream of Eδ in the murine Tcra-Tcrd locus. The ectopic CBE impaired inversional rearrangement of Trdv5 in the absence of measurable effects on Trdv5 transcription and chromatin accessibility. Moreover, although the ectopic CBE limited directional RAG tracking from the Tcrd recombination center, such tracking cannot account for Trdv5-to-Trdd2 inversional rearrangement. Rather, the defect in Trdv5 rearrangement could only be attributed to a reconfigured chromatin loop organization that limited RAG-independent through-space interactions between the Trdv5 and Trdd2 RSSs. We conclude that CTCF can regulate V(D)J recombination by segregating RSSs into distinct loop domains and inhibiting RSS synapsis, independent of any effects on transcription, RSS accessibility and RAG tracking.
2016-09-02 | GSE81351 | GEO
Project description:In developing B lymphocytes, V(D)J recombination assembles IgH and Igk variable region exons from hundreds of gene segments clustered across mega-base Igh and Igk loci. V, D, and J segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination center (RC). JH-RSS orientation programs RAG to scan upstream D- and VH-containing chromatin linearly presented by cohesin-mediated loop extrusion. During Igh scanning, RAG robustly utilizes only D- or VH-RSSs in convergent ("deletional") orientation with JH-RSSs. However, for Vk-to-Jk joining, RAG utilizes Vk-RSSs from deletional- and inversional-oriented clusters, inconsistent with linear scanning. Here, we elucidate the Vk-to-Jk joining mechanism. Igk undergoes robust primary and secondary rearrangements, which confounds scanning assays. Thus, we engineered cells to undergo only primary Vk-to-Jk rearrangements and found that RAG-scanning from the primary Jk-RC terminates just 8kb upstream within the CTCF-Site-based Sis element. While Sis and the Jk-RC barely interacted with the Vk locus, the CTCF-site-based Cer element, 4kb upstream of Sis, interacted with various loop-extrusion impediments across the locus. Like VH-locus inversion, DJH inversion abrogated VH-to-DJH joining; yet Vk-locus or Jk inversion allowed robust Vk-to-Jk joining. Together, these experiments implicated loop extrusion in bringing Vks near Cer for short-range diffusion-mediated capture by RC-based RAG. To elucidate key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vk-to-JH and D-to-Jk rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vk and Jk RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed ability of Igk versus Igh RSSs to promote robust diffusional joining. We propose that Igk evolved strong RSSs to mediate diffusional Vk-to-Jk joining; while Igh evolved weaker RSSs requisite for modulating VH joining by RAG scanning impediments.
2024-04-08 | GSE254039 | GEO
Project description:In developing B lymphocytes, V(D)J recombination assembles IgH and Igk variable region exons from hundreds of gene segments clustered across mega-base Igh and Igk loci. V, D, and J segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination center (RC). JH-RSS orientation programs RAG to scan upstream D- and VH-containing chromatin linearly presented by cohesin-mediated loop extrusion. During Igh scanning, RAG robustly utilizes only D- or VH-RSSs in convergent ("deletional") orientation with JH-RSSs. However, for Vk-to-Jk joining, RAG utilizes Vk-RSSs from deletional- and inversional-oriented clusters, inconsistent with linear scanning. Here, we elucidate the Vk-to-Jk joining mechanism. Igk undergoes robust primary and secondary rearrangements, which confounds scanning assays. Thus, we engineered cells to undergo only primary Vk-to-Jk rearrangements and found that RAG-scanning from the primary Jk-RC terminates just 8kb upstream within the CTCF-Site-based Sis element. While Sis and the Jk-RC barely interacted with the Vk locus, the CTCF-site-based Cer element, 4kb upstream of Sis, interacted with various loop-extrusion impediments across the locus. Like VH-locus inversion, DJH inversion abrogated VH-to-DJH joining; yet Vk-locus or Jk inversion allowed robust Vk-to-Jk joining. Together, these experiments implicated loop extrusion in bringing Vks near Cer for short-range diffusion-mediated capture by RC-based RAG. To elucidate key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vk-to-JH and D-to-Jk rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vk and Jk RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed ability of Igk versus Igh RSSs to promote robust diffusional joining. We propose that Igk evolved strong RSSs to mediate diffusional Vk-to-Jk joining; while Igh evolved weaker RSSs requisite for modulating VH joining by RAG scanning impediments.
2024-04-08 | GSE263123 | GEO
Project description:The large antigen receptor (AgR) loci in T and B lymphocytes have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the far end of each locus near enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus which also shows significant lineage-specificity (T vs. B cells) and developmental stage-specificity (pre-B, not pro-B) in CTCF binding. Cohesin is often bound at the same sites as CTCF, and cohesin is thought to create long range chromatin contacts by loop extrusion, with its translocation stopped by convergently oriented CTCF sites. Importantly, cohesin binding shows greater lineage- and stage- specificity than CTCF at most loci, thus providing more specificity to the CTCF/cohesin loops in AgR loci. Since all the CTCF sites within the large V portions of the Igh and TCRβ loci have the same orientation, this suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D‐J‐enhancer end of each locus. The V region portions of the Igκ and TCRα δ loci, in contrast, have CTCF sites in both orientations, providing many options for creating CTCF-mediated loops throughout the loci. The immune system may have developed unique utilization of CTCF sites to generate lymphocyte-specific long-range loops to facilitate the formation of diverse antigen receptor repertoires.
2018-04-18 | GSE109909 | GEO
Project description:The Tcra/Tcrd locus undergoes V(D)J recombination in CD4−CD8− double-negative (DN) thymocytes and CD4+CD8+ double-positive (DP) thymocytes to generate diverse TCRδ and TCRα repertoires, respectively. Here we reveal a Tcra/Tcrd locus chromatin interaction network in DN thymocytes that is formed by interactions between CTCF-binding elements (CBEs). Disruption of a discrete chromatin loop encompassing the Dδ, Jδ and Cδ gene segments allows a single Vδ segment to frequently contact and rearrange to Dδ and Jδ segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrows the TCRα repertoire, which, we believe, follows as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. Examination of chromatin loops by 4C-seq from 4 viewpoints in two lymphoid cell compartments: CD4-CD8- thymocytes and naïve B splenocytes.
2015-08-10 | E-GEOD-67442 | biostudies-arrayexpress
Project description:RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RC from upstream VHs in a chromatin loop anchored by CTCF-binding elements ("CBEs"). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and, thereby, promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
2018-07-03 | GSE113022 | GEO
Project description:RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RC from upstream VHs in a chromatin loop anchored by CTCF-binding elements ("CBEs"). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and, thereby, promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
2018-07-03 | GSE112822 | GEO
Project description:RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RC from upstream VHs in a chromatin loop anchored by CTCF-binding elements ("CBEs"). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and, thereby, promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
2018-07-03 | GSE112781 | GEO
Project description:Immunoglobulin heavy chain locus (Igh) VH, D, and JH gene segments are developmentally assembled into V(D)J exons. RAG endonuclease initiates V(D)J recombination by binding a JH-recombination signal sequence (RSS) within a chromatin-based recombination center (RC) and then, in an orientation-dependent process, scans upstream D-containing chromatin presented by cohesin-mediated loop extrusion for convergent D-RSSs to initiate DJH-RC formation. In primary pro-B cells, 100s of upstream VH-associated RSSs, embedded in convergent orientation to the DJH-RC-RSS, gain proximity to the DJH-RC for VH-to-DJH joining via a mechanistically-undefined VH-locus contraction process. Here, we report that a 2.4 mega-base VH locus inversion in primary pro-B cells nearly abrogates rearrangements of normally convergent VH-RSSs and cryptic RSSs, even though locus contraction per se is maintained. Moreover, this inversion activated rearrangement of both cryptic VH-locus RSSs normally in the opposite orientation and, unexpectedly, of normally-oriented cryptic RSSs within multiple, sequential upstream convergent-CBE domains. Primary pro-B cells had significantly reduced transcription of Wapl, a cohesin-unloading factor, versus levels in v-Abl pro-B lines that lack marked locus contraction or distal VH rearrangements. Correspondingly, Wapl depletion in v-Abl lines activated VH-locus contraction and orientation-specific RAG-scanning across the VH-locus. Our findings indicate that locus contraction and physiological VH-to-DJH joining both are regulated via circumvention of CBE scanning impediments.
2020-11-08 | GSE151906 | GEO